Anti-obesity and anti-dyslipidemic effects of oil palm phenolics in treating atherosclerosis and cardiovascular disease

ABSTRACT

A composition comprising extracts containing oil palm phenolics in an amount effective for use in a method of reducing cholesterol biosynthesis and thus preventing obesity. The composition up regulates fatty acid beta oxidation and down regulates cholesterol biosynthesis in livers. The composition is useful for prevention of obesity associated diseases. The composition delays the onset of obesity and attenuates the inflammatory response of atherogenic diet, whereby the composition aids to suppress the inflammatory response thereby ameliorating artherosclerosis. The composition delays weight gain or obesity thereby preventing the effects of dyslipidemia.

CLAIM OF PRIORITY

This application is a continuation of U.S. application Ser. No.13/521,055, filed on Mar. 15, 2013, which is a National Phaseapplication of International Application No.: PCT/MY2011/000002, filedon Jan. 7, 2011, which claims priority to Malaysian Patent ApplicationNo. PI2010000060, filed on Jan. 7, 2010, which are hereby incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The present invention provides a method of delaying weight gain orobesity and preventing the effects of dyslipidemia caused by anatherogenic diet in an animal. It comprises the step of administeringoil palm phenolics in the drinking water to said animal. Theaforementioned properties of oil palm phenolics are attributed to theup-regulation of fatty acid beta oxidation and down-regulation ofcholesterol biosynthesis in the liver, when the said animal was given alow-fat normal diet. In addition, oil palm phenolics also up-regulatedunfolded protein response in the liver, down-regulated antigenpresentation and processing in the spleen as well as up-regulatedantioxidant genes in the heart, when the said animal was given ahigh-fat, cholesterol-containing, atherogenic diet. Oil palm phenolicsmay thus confer anti-inflammatory and antioxidative effects for theprevention of atherosclerosis and cardiovascular disease.

BACKGROUND OF THE INVENTION

Cardiovascular disease, together with atherosclerosis, is the firstcause of death in the world. A variety of risk factors are known to beassociated with the pathogenesis of atherosclerosis and cardiovasculardisease. These factors include hypercholesterolemia, hyperlipidemia,hyperglycemia, hypertension, obesity, elevated levels of plasmahomocysteine and hemostatic factors, family history, the male gender,stress, smoking, lack of exercise, high-fat diets, infectious agents andaging. People with diabetes usually have more severe debility fromatherosclerotic events over time than non-diabetics.

Atherosclerosis is a disease affecting arterial blood vessels, and itinvolves the hardening (calcification) of arteries and the formation ofatheromatous plaques within the arteries. As with other chronicdiseases, atherosclerosis is believed to be caused by the accumulationof harmful free radicals and reactive oxygen species in the body. It isassociated with systemic immune responses and inflammation.Atherosclerosis causes two main problems, infarction (complete coronaryocclusion) and aneurysm (partial coronary occlusion), and it canactually be viewed as a problem of wound healing and chronicinflammation. Atheroslerosis may cause brain strokes, heart attacks andperipheral artery occlusive diseases in the lower extremities.

The pathophysiology of atherosclerosis comprises various importantsteps, including enhanced endothelial focal adhesiveness, permeabilityand pro-coagulation (endothelial dysfunction), expression of adhesionmolecules, monocyte adhesion and immigration, formation of foam cell andfatty streaks, smooth muscle cell (SMC) migration from the tunica mediainto the tunica intima, plaque formation and finally, plaque rupture andthrombus formation. A prevalent theme in atherosclerosis is thus thepresence of oxidative stress and inflammation, due to the oxidation ofLDL.

The oxidation of low-density lipoprotein (LDL) has been accepted as animportant initial event in the development of atherosclerosis. Reactiveoxygen species can stimulate the oxidation of LDL, and oxidized LDLwhich is not recognized by the LDL receptor is then taken up byscavenger receptors in macrophages leading to foam cell formation andatheromatous plaques. In addition, macrophages also possess toll-likereceptors which bind pathogen-like molecules and initiate a signallingcascade which leads to cell activation. These macrophages produceinflammatory cytokines, chemokines, free radicals, growth-regulatingmolecules, metalloproteinases and other hydrolytic enzymes. Apoptosis offoam cells, which is influenced by cytokine expression and themacrophage activation state also contributes to the formation of anecrotic core.

Previous studies show that among the genes which have increasedexpression in the atherosclerotic vessel wall are those involved ininflammation, such as chemokine and chemokine receptors, interleukin andinterleukin receptors, major histocompatibility complex (MHC) molecules,endothelial cell adhesion molecules, extracellular matrix and matrixremodeling proteins, matrix metalloproteinase genes, transcriptionfactors, lipid metabolism and vascular calcification genes, as well asmacrophages and smooth muscle cell specific genes. On the other hand,among the genes with decreased expression in the atherosclerotic vesselwall include anti-adhesive, anti-proliferative and anti-inflammatorygenes as well as differentiated muscle markers.

Besides surgical interventions such as angioplasty and bypass surgery,various pharmacological interventions have been used for the treatmentof atherosclerosis and the associated cardiovascular disease.Medications to lower cholesterol and LDL as well as those which increasehigh-density lipoprotein (HDL) are normally utilized to prevent theoccurrence of atherosclerosis. For example, statins are used to inhibitan enzyme called Hmgcr (3-hydroxy-3-methylglutaryl-coenzyme-Areductase), which is involved in cholesterol biosynthesis. Yet anothertherapeutic strategy in the treatment of atherosclerosis is the use ofcell cycle inhibitors which include pharmacological agents, irradiationor gene therapy, as vascular proliferation is central toatherosclerosis.

Immunosuppressive and anti-inflammatory drugs such as cyclosporine whichblock the activation of T cells may also be used as a therapeutictreatment for atherosclerosis. In addition to its cholesterol-loweringproperties, statins also show pleiotropic effects includingimmunosuppressive properties. Vaccination with oxidized LDL, bacteriacontaining modified phospholipids or heat shock proteins is also anattractive approach to induce protective immunity againstatherosclerosis. Yet other approaches include transfer ofanti-inflammatory interleukins and administration of decoys andantibodies directed against pro-inflammatory interleukins.

Most of the current approaches however, aim to treat atherosclerosisrather than to prevent it. With the increase in health awareness amongthe public, it was realized through epidemiological and experimentalstudies that diets containing high amount of phytochemicals can alsoprovide protection against free radical-induced diseases such asatherosclerosis and cardiovascular disease, due to their highantioxidant activities. For example, dietary antioxidants such asvitamin E, vitamin C, carotenoids, polyphenols and coenzyme Q10 werefound to be able to prevent atherogenesis.

Phenolic antioxidants from soy, pomegranate, ginger and red wine werealso found to attenuate atherosclerosis either by LDL-dependentmechanisms such as reducing LDL levels, inhibiting LDL oxidation andincreasing the antioxidant status or via other LDL-independentmechanisms. Resveratrol, a phenolic phytoalexin found in red wine, wasalso suggested to mediate cardioprotection through the preconditioningeffect, rather than direct protection. Preconditioning is achieved bysubjecting the heart to a therapeutic amount of stress, therebydisturbing normal cardiovascular homeostasis and reestablishing amodified homeostatic condition with increased cardiac defences that canwithstand subsequent stress insults. Resveratrol was also found toincrease the lifespan and survival of mice on a high-calorie diet. Plantphenolics are thus promising candidates for the prevention ofatherosclerosis and related cardiovascular disease.

The oil palm (Elaeis guineensis) contains various phytochemicals whichpossess significant antioxidant properties such as carotenoids,tocopherols and tocotrienols. The extraction of water-soluble phenolicsfrom the palm oil mill effluent (POME) through a completely solvent-freeprocess recovers another type of antioxidant from the oil palm,designated the Essence of Palm® which contains various phenolic acidsand polyphenols. This discovery potentiates the two-pronged approach ofreducing environment pollution caused by POME while producing premiumproducts for the pharmaceutical, nutraceutical and cosmeceuticalmarkets. Oil palm phenolics showed significant biological activitiesagainst LDL oxidation, increased the amounts of HDL in hamsters fed anatherogenic diet and attenuated atherosclerosis in blood vessels ofatherogenic diet-fed rabbits.

In this study, we extended the knowledge that oil palm phenolics canattenuate atherosclerosis by hypothesizing that the extract mightinfluence certain gene expression changes. We thus tested thishypothesis by feeding mice with either a low-fat normal diet (14.6%kcal/kcal energy) or a high-fat (40.5% kcal/kcal energy) atherogenicdiet containing cholesterol (0.15% w/w). Each group was further giveneither distilled water (control group) or oil palm phenolics (treatmentgroup). By harvesting major organs such as livers, spleens and heartsfor microarray gene expression profiling analysis, we identified thebiological changes caused by oil palm phenolics in the normal diet fedmice, by the atherogenic diet and by oil palm phenolics in theatherogenic diet fed mice, as well as discovered how the extract changedthe gene expression profiles caused by the atherogenic diet.

SUMMARY OF THE INVENTION

The invention relates to a composition useful for providing anti-obesityor anti-dyslipidemics properties, and thus the prevention ofartherosclerosis and cardiovascular diseases related thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a graph for body weights of mice, and FIG. 1B shows agraph of organ weights of mice treated with (1) normal diet+distilledwater; (2) normal diet+oil palm phenolics; (3) atherogenicdiet+distilled water; and (4) atherogenic diet+oil palm phenolics. Thedata points marked with # represent p<0.05 vs. Normal Diet+DistilledWater; n=10. Error bars indicate S.E.M.

FIG. 2A depicts the GenMAPPs showing functions and genes significantlyup-regulated in the liver fatty acid beta oxidation pathway by oil palmphenolics, and FIG. 2B depicts the GenMAPPs showing functions and genessignificantly down-regulated in the liver cholesterol biosynthesispathway by oil palm phenolics.

FIG. 3A shows that the genes up-regulated by the Atherogenic Diet in theliver are linked to Hnf4a, which is a nuclear factor involved inhepatocyte differentiation; and FIG. 3B shows that the up-regulatedgenes include cytochrome c oxidases, caspases and complement genes,which are involved in cell death via apoptosis.

FIG. 4 is a diagram of genes down-regulated by the Atherogenic Diet inthe Liver Cholesterol Biosynthesis Pathway;

FIG. 5A is a diagram of genes up-regulated by the Atherogenic Diet inthe Stat3 network (e.g., in the Spleen); and FIG. 5B is a diagram ofgenes down-regulated in the Tp53 network.

FIG. 6A is a diagram of genes up-regulated by the Atherogenic Diet inthe Jun network (e.g., in the heart); and FIG. 6B is a diagram of genesdown-regulated in the Tgfb1 network.

FIG. 7 is a diagram of genes up-regulated by oil palm phenolics in theliver unfolded protein response network;

FIG. 8 is a diagram of genes down-regulated by oil palm phenolics in thespleen antigen presentation network;

FIG. 9 is a diagram of genes up-regulated by oil palm phenolics in theheart antioxidant pathway;

FIG. 10 is a diagram of the percentage of genes which showed a change indirection when regulated by oil palm phenolics as compared to theatherogenic diet;

FIGS. 11A and 11B show a comparison of genes that are significantlychanged by the atherogenic diet and oil palm phenolics in terms of thedirection of fold changes (liver data as example);

FIG. 12A is a diagram of the direction and magnitude of gene expressionfold changes of eight target genes as determined by microarray andreal-time qRT-PCR experiments and their correlation; the direction andmagnitude of fold changes obtained from the real-time qRT-PCR techniquewere comparable to those obtained from the microarray technique. *P<0.05 for gene expression fold changes quantitated by real-time PCRexperiments as determined by two-tailed unpaired Student's t-test.

FIG. 12B presents a validation of the microarray data via real-timeqrt-PCR and shows that correlation of fold changes obtained by these twogene expression profiling techniques was high with an R²=0.9877.

FIG. 13A diagrams results of cytokine profiling on blood serum samplesfrom mice, based on the original Y-axis; and FIG. 13B diagrams resultsof cytokine profiling on blood samples from mice, based an adjustedY-axis. # denotes those samples for which p<0.05; n=6. Error barsindicate S.E.M.

FIGS. 14A, 14B, 14C and 14D collectively diagram results of antioxidantanalysis on blood serum samples from mice; FIG. 14A relates specificallyto TP-FCR; FIG. 14B relates specifically to FRAP; FIG. 14C relatesspecifically to DPPH; and FIG. 14D relates specifically to TEAC. #identifies those samples for which p<0.05 vs. Normal Diet+DistilledWater; n=6. Error bars indicate S.E.M.

DETAILED DESCRIPTION

All male inbred BALB/c mice (n=40) which were designated for this studywere purchased from the Institute of Medical Research, Kuala Lumpur,Malaysia, at around five weeks of age just after weaning. All animalprocedures were approved by the Animal Care and Use Committee of theUniversity of Malaya, Kuala Lumpur, Malaysia. The animals were randomlyassigned into cages (n=5 per cage) and acclimatized for one week, duringwhich a standard chow diet purchased from the University of Malaya, anddistilled water were given. At the start of the experiment, the diet ofthe animals was changed to a custom-made low-fat normal diet (58.2%kcal/kcal carbohydrate, 27.2% kcal/kcal protein and 14.6% kcal/kcal fat,including cellulose, mineral mix, vitamin mix and DL-methonine) or acustom-made high-fat atherogenic diet (40.5% kcal/kcal carbohydrate,19.0% kcal/kcal protein and 40.5% kcal/kcal fat, including 0.15% w/wcholesterol, as well as cellulose, mineral mix, vitamin mix andDL-methonine). The normal control group (n=10) and the atherogeniccontrol group (n=10) were supplemented with distilled water while thenormal treatment group (n=10) and the atherogenic treatment group (n=10)were supplemented with oil palm phenolics as drinks ad libitum. Theantioxidant content of the oil palm phenolics given was around 1500 ppmgallic acid equivalent. Food and fluid were changed daily. During theanimal feeding process, body weights were monitored every week. Aftersix weeks, the mice were sacrificed via euthanasia with diethyl etherand blood samples were collected via cardiac puncture. Six major organsincluding livers, spleens, hearts, kidneys, lungs and brains wereexcised, blotted, weighed, snap-frozen in liquid nitrogen and stored at−80° C.

The body weights of mice steadily increased every week throughout thesix weeks of feeding, with those on the atherogenic diet showing ahigher increase in weight gain compared to those on the normal diet(FIG. 1A). In contrast, mice in the normal treatment group showed adelay in weight gain throughout the six weeks of feeding (FIG. 1A). Whenthe organ weights from the animals were compared, the atherogenic dietwas found to cause an increase in the weight of mouse livers (FIG. 1B).On the other hand, oil palm phenolics did not significantly affect organweights, both in mice given the normal diet and the atherogenic diet(FIG. 1B). These results indicate that the addition of extra fat andcholesterol in the atherogenic diet increased its energy content. Thiscaused the livers of mice in the two atherogenic diet groups to enlargein order to accommodate an increased need for fat and cholesterolprocessing, and resulted in a higher weight gain.

A portion (200 μL) of whole blood samples obtained from about half ofthe animals (n=4) was aliquoted into a tube containingethylenediaminetetraacetic acid (EDTA) (Ambion, Austin, Tex.) to preventclotting. These whole blood samples were immediately sent afterdissection of the animals to the Clinical Biochemistry and HematologyLaboratory, Department of Veterinary Pathology and Microbiology, Facultyof Veterinary Medicine, University of Putra Malaysia (UPM), Serdang,Selangor, Malaysia, for hematology analysis. The analysis was carriedout using the Animal Blood Counter Vet Hematology Analyzer (Horiba ABX,France).

In order to obtain sera, the remaining blood samples from all of theanimals (n=10) were allowed to clot at room temperature for 2 hoursbefore centrifuging them at 3300 rpm for 5 minutes, after which thesupernatant layers were collected and stored at −20° C. A portion (100μL) of each serum sample (n=6 per group) was kept in aliquots forcytokine profiling and antioxidant analysis. The remaining serum samples(around 200 μL per replicate) were then sent for clinical biochemistryanalysis using the Roche/Hitachi 902 Chemistry Analyzer (Roche/Hitachi,Switzerland) in the Clinical Biochemistry and Hematology Laboratory,Department of Veterinary Pathology and Microbiology, Faculty ofVeterinary Medicine, UPM. Clinical biochemistry parameters which wereexamined include alanine aminotransferase, aspartate aminotransferase,glucose, serum total protein, albumin, globulin, albumin:globulin ratio,total cholesterol, triglycerides, low-density lipoprotein (LDL) andhigh-density lipoprotein (HDL). Two samples in each control group andthree samples in each treatment group were excluded from data analysisdue to blood lysis.

In terms of hematology, mice given the atherogenic diet showed asignificant increase in the levels of white blood cells, neutrophils andlymphocytes when compared to those given the normal diet (Table 1),indicating the presence of an inflammatory response. Oil palm phenolicsdid not affect hematology parameters in both modules (Table 1). In termsof clinical biochemistry, significant changes caused by the atherogenicdiet involved the levels of glucose (⬇), albumin (⬇), globulin (⬆), A:G(⬇), total cholesterol (⬆), LDL-C(⬆) and HDL-C(⬆) (Table 2). Oil palmphenolics did not cause significant changes in the clinical biochemistryparameters measured in each module, except for normalizing glucoselevels in the atherogenic diet module (Table 2).

TABLE 1 Hematology Parameters Measured Using Mouse Whole Blood SamplesNormal Diet + Normal Diet + Atherogenic Diet + Atherogenic Diet + TestDistilled Water Oil Palm Phenolics Distilled Water Oil Palm PhenolicsRed Blood Cells (×10¹²/L) 9.93 ± 0.32 ^(a) 10.15 ± 0.12 ^(a)  10.08 ±0.21 ^(a) 10.02 ± 0.08 ^(a) Hemoglobin (g/L) 148 ± 4 ^(a)  149 ± 1 ^(a) 149 ± 3 ^(a)  149 ± 1 ^(a)  Hematocrit/Packed Cell Volume (L/L) 0.40 ±0.01 ^(a) 0.40 ± 0.00 ^(a)  0.40 ± 0.02 ^(a)  0.40 ± 0.00 ^(a) MeanCorpuscular Volume (fL) 41 ± 1 ^(a)  40 ± 0 ^(a)  40 ± 1 ^(a) 40 ± 1^(a) Mean Corpuscular Hemoglobin 369 ± 6 ^(a)  373 ± 4 ^(a)  373 ± 7^(a)  372 ± 5 ^(a)  Concentration (g/L) White Blood Cells (×10⁹/L) 2.0 ±0.6 ^(a) 1.5 ± 0.3 ^(a)  3.3 ± 0.3 ^(b)  3.0 ± 0.1 ^(b) B Neutrophils(×10⁹/L) 0.05 ± 0.01 ^(a) 0.04 ± 0.01 ^(a)  0.10 ± 0.02 ^(b)  0.10 ±0.02 ^(b) S Neutrophils (×10⁹/L) 0.48 ± 0.17 ^(a) 0.35 ± 0.09 ^(a)  0.83± 0.07 ^(b)  0.77 ± 0.06 ^(b) Lymphocytes (×10⁹/L) 1.36 ± 0.38 ^(a) 1.01± 0.20 ^(a)  2.06 ± 0.22 ^(b)  1.99 ± 0.17 ^(b) Monocytes (×10⁹/L) 0.09± 0.02 ^(a) 0.07 ± 0.02 ^(a)  0.08 ± 0.04 ^(a)  0.09 ± 0.03 ^(a)Eosinophils (×10⁹/L) 0.03 ± 0.01 ^(a) 0.01 ± 0.00 ^(a)  0.06 ± 0.02 ^(a) 0.06 ± 0.03 ^(a) Basophils (×10⁹/L) 0.00 ± 0.00 ^(a) 0.00 ± 0.00 ^(a) 0.00 ± 0.00 ^(a)  0.00 ± 0.00 ^(a) Thrombocytes (×10⁹/L) 533 ± 111 ^(a)621 ± 103 ^(a) 644 ± 37 ^(a) 619 ± 21 ^(a) P Prothrombin (g/L) 79 ± 2^(a)  80 ± 1 ^(a)  78 ± 2 ^(a) 79 ± 2 ^(a) Values shown are Means ±S.E.M.; Means with different superscript letters are significantlydifferent (P < 0.05).

TABLE 2 Clinical Biochemistry Parameters Measured Using Mouse SerumSamples Normal Diet + Normal Diet + Atherogenic Diet + AtherogenicDiet + Test Distilled Water Oil Palm Phenolics Distilled Water Oil PalmPhenolics Alanine Aminotransferase (ALT) (U/L) 34.4 ± 3.3 ^(a)  42.5 ±6.5 ^(a)  41.8 ± 10.7 ^(a) 32.2 ± 5.1 ^(a)  Aspartate Aminotransferase(AST) (U/L) 175.2 ± 23.8 ^(a)  240.4 ± 22.3 ^(a)  174.8 ± 29.3 ^(a) 157.2 ± 32.2 ^(a)  Glucose (mmol/L) 6.0 ± 1.1 ^(a) 6.7 ± 0.4 ^(a)  5.3 ±0.4 ^(a,b)  7.4 ± 0.4 ^(a,c) Serum Total Protein (g/L) 53.8 ± 1.8 ^(a) 53.8 ± 1.1 ^(a)  53.2 ± 0.9 ^(a)  54.8 ± 0.7 ^(a)  Albumin (g/L) 34.0 ±0.9 ^(a)  33.1 ± 1.3 ^(a)  29.4 ± 0.7 ^(b)  31.0 ± 0.7 ^(b)  Globulin(g/L) 19.8 ± 1.1 ^(a)  20.8 ± 0.8 ^(a)  23.8 ± 0.7 ^(b)  23.7 ± 0.7^(b)  A:G 1.8 ± 0.1 ^(a) 1.6 ± 0.1 ^(a) 1.2 ± 0.1 ^(b) 1.3 ± 0.1 ^(b)Total Cholesterol (mmol/L) 3.46 ± 0.13 ^(a) 3.53 ± 0.19 ^(a) 4.77 ± 0.15^(b) 4.76 ± 0.19 ^(b) Triglycerides (mmol/L) 1.05 ± 0.08 ^(a) 1.04 ±0.11 ^(a) 1.13 ± 0.04 ^(a) 1.14 ± 0.15 ^(a) Low-Density Lipoprotein(mmol/L) 0.15 ± 0.02 ^(a) 0.18 ± 0.03 ^(a) 0.26 ± 0.03 ^(b) 0.30 ± 0.06^(b) High-Density Lipoprotein (mmol/L) 2.79 ± 0.11 ^(a) 2.83 ± 0.17 ^(a)4.05 ± 0.11 ^(b) 3.93 ± 0.14 ^(b) Values shown are Means ± S.E.M.; Meanswith different superscript letters are significantly different (P <0.05).Microarray Gene Expression Analysis

For gene expression analysis, livers from the normal diet module as wellas livers, spleens and hearts from the atherogenic diet module were usedin the total RNA extraction process. Total RNA isolation from mouseorgans was carried out using the RNeasy Mini Kit (Qiagen, Inc.,Valencia, Calif.) and QIAshredder homogenizer (Qiagen, Inc., Valencia,Calif.). The total RNA samples obtained were subjected to NanoDrop 1000ASpectrophotometer for yield and purity assessment. Integrity of thetotal RNA samples was then assessed using the Agilent 2100 Bioanalyzer(Agilent Technologies, Palo Alto, Calif.) and Agilent RNA 6000 Nano ChipAssay Kit (Agilent Technologies, Palo Alto, Calif.). Four total RNAsamples with the highest RNA Integrity Numbers and 28S/18S rRNA ratioswithin each condition were then selected for microarray studies.

Amplification of total RNA samples which were of high yield, purity andintegrity was carried out using the Illumina TotalPrep RNA AmplificationKit (Ambion, Inc., Austin, Tex.). The cRNA produced was then hybridizedto the Illumina MouseRef-8 Expression BeadChip Version 1 (Illumina,Inc., San Diego, Calif.), using the Direct Hybridization Kit (Illumina,Inc., San Diego, Calif.). Illumina MouseRef-8 Expression BeadChipscontained 50-mer gene-specific probes for over 24000 genes which weredesigned based on the Mouse Exonic Evidence Based Oligonucleotide(MEEBO) set, the RIKEN FANTOM 2 database and the National Center forBiotechnology Information (NCBI) RefSeq (Release 5) transcript database.Microarray hybridization, washing and scanning were carried outaccording to the manufacturer's instructions.

In brief, cRNA was added with a hybridization buffer, and thehybridization mixture was then briefly heated and hybridized to anIllumina BeadChip. The hybridized microarray then underwent a series ofwashes using the wash buffers provided and 100% ethanol (Merck,Darmstadt, Germany). Non-specific hybridization was blocked beforeincubating the microarray with the Amersham Fluorolink Streptavidin Cy-3dye (GE Healthcare Bio-Sciences, Little Chalfont, UK) for detection,followed by a final wash with the wash buffer. The microarray was thendried and scanned with the Illumina BeadArray Reader confocal scannerand Illumina BeadScan software (Illumina, Inc., San Diego, Calif.),available at the Malaysia Genome Institute, National University ofMalaysia.

Quality control of the hybridization, microarray data extraction andinitial analysis were carried out using the Illumina BeadStudio software(Illumina, Inc., San Diego, Calif.). Outlier samples were removed viahierarchical clustering analysis provided by the Illumina BeadStudiosoftware and also using the TIGR MeV software, via different distancemetrics. A minimum of three replicates per condition (with outliersremoved) was then considered for further analysis.

It should be noted that four comparisons of the microarray data obtainedwere made in this study, with the first comparison to find out geneexpression changes caused by oil palm phenolics in the normal dietmodule (Normal Diet+Oil Palm Phenolics: Normal Diet+Distilled Water).The second comparison was made to find out gene expression changescaused by the atherogenic diet (Atherogenic Diet+Distilled Water: NormalDiet+Distilled Water). The third comparison was made to identify geneexpression changes caused by oil palm phenolics in the atherogenic dietmodule (Atherogenic Diet+Oil Palm Phenolics: Atherogenic Diet+DistilledWater). The fourth comparison was carried out to identify genes whichwere regulated differently by the atherogenic diet and oil palmphenolics, by comparing results from the second and third comparison.The first three comparisons were carried out separately before thefourth comparison was made.

For the first three comparisons, gene expression values were normalizedusing the rank invariant method and genes which had a Detection Level ofmore than 0.99 in either condition (control or treatment) wereconsidered significantly detected. To filter the data for genes whichchanged significantly in terms of statistics, the Illumina Custom errormodel was used and genes were considered significantly changed at a|Differential Score| of more than 20, which was equivalent to a P Valueof less than 0.01. The stringency of this filtering criterion waslowered to a |Differential Score| of more than 13, which was equivalentto a P Value of less than 0.05, should less than 100 genes wereconsidered significantly changed. Since the results of this statisticalanalysis would be used for functional analysis, it would be relevant toinclude more genes by using a lower threshold to give statistical powerto the functional analysis, in which functional significance could beassessed.

The genes and their corresponding data were then exported into theMicrosoft Excel software for further analysis. To calculate foldchanges, an arbitrary value of 10 was given to expression values whichwere less than 10. Fold changes were then calculated by dividing meansof Signal Y (treatment) with means of Signal X (control) if the geneswere up-regulated and vice versa if the genes were down-regulated.Two-way (gene and sample) hierarchical clustering of the significantgenes was then performed using the TIGR MeV software to ensure that thereplicates of each condition were clustered to each other. The Euclideandistance metric and average linkage method were used to carry out thehierarchical clustering analysis.

For the first three comparisons, changes in biological pathways and geneontologies were also assessed via functional analysis, using the GenMAPPand MAPPFinder softwares. The MAPPFinder software ranks GenMAPPs(pathways) and gene ontologies based on hypergeometric distribution.GenMAPPs and gene ontologies which had Permuted P Values of less than0.01, Numbers of Genes Changed of more than or equal to 2 and Z Scoresof more than 2 were considered significant. A Permuted P Value of lessthan 0.05 was used when genes were selected using a |Differential Score|of more than 13, in order to identify more GenMAPPs and gene ontologiesaffected.

It should be noted that the MAPPFinder software clusters multiple probesfor a distinct gene into a single gene grouping in order to calculatethe number of distinct genes which meet the user-defined criteria, notthe probes. In this study, up- and down-regulated genes were analyzedseparately in the functional enrichment analysis but were viewedtogether in each GenMAPP. Boxes coloured yellow indicate genes whichwere up-regulated while those coloured blue indicate genes which weredown-regulated. The fold changes are indicated next to the boxes.Individual boxes which have different shadings within them indicate thepresence of multiple probes (splice transcripts) within a single gene.

Changes in regulatory networks were also analyzed through the use ofIngenuity Pathways Analysis software (Ingenuity® Systems, Redwood City,Calif.) [36] for the first three comparisons. A data set containingdifferentially expressed genes and their corresponding fold changes wasuploaded into the application. Analysis of up- and down-regulated geneswere carried out separately. Each gene identifier was mapped to itscorresponding gene object in the Ingenuity Pathways Knowledge Base.These genes were overlaid onto a global molecular network developed frominformation contained in the Ingenuity Pathways Knowledge Base. Networksof these focus genes were then algorithmically generated based on theirconnectivity.

A network is a graphical representation of the molecular relationshipsbetween genes or gene products. Genes or gene products were representedas nodes, and the biological relationship between two nodes wasrepresented as an edge (line). The intensity of the node color indicatesthe degree of up- (red) or down- (green) regulation. Nodes weredisplayed using various shapes that represented the functional class ofthe gene product. Edges were displayed with various labels thatdescribed the nature of the relationship between the nodes. Genedescriptions which were not referenced emanated directly from theIngenuity Pathways Analysis software.

Oil Palm Phenolics Up-Regulated Fatty Acid Beta Oxidation Genes andDown-Regulated Cholesterol Biosynthesis Genes in the Liver (Normal DietModule)

Oil palm phenolics up-regulated 196 genes and down-regulated 54 genes inthe livers of mice on a normal diet, with the lists of genes andfunctions significantly changed supplemented in Additional Files 1 and 2respectively. Functional analysis on the microarray data from the livershowed that oil palm phenolics up-regulated the fatty acid betaoxidation pathway (FIG. 2A). Among the fatty acid beta oxidation genesup-regulated were those encoding sterol carrier protein (Scp),lysophospholipase (Lypla1), monoglyceride lipase (Mgll), acetyl-coAdehydrogenase (Acadl), acyl-coA dehydrogenases (Acads, Acad8),hydroxyacyl-coA dehydrogenases (Hadhb, Hadhsc), acetyl-coAacetyltransferases (Acat2, Acat3) and acetyl-coA acyltransferase(Acaa2).

The liver is known as an organ active in fatty acid beta oxidation, andthus up-regulation of hepatic fatty acid beta oxidation might contributeto the suppression of liver fat and visceral fat accumulation.Up-regulated fatty acid beta oxidation may also contribute to theprevention of diabetes, which is known to be caused by obesity andinsulin resistance. Up-regulation of genes involved in lipid catabolismhas also been found to be caused by the catechins of green tea and thechlorogenic acid of coffee. In addition, removal of lipids from the bodythrough fatty acid beta oxidation may prevent lipid peroxidation, whichcontributes to atherosclerosis. Interestingly, enhanced hepatic fattyacid synthesis and reduced fatty acid oxidation have also been impliedin the development of an alcohol-induced fatty liver. Thus, we postulatethat oil palm phenolics may also be able to prevent alcohol-inducedliver damage by up-regulating hepatic fatty acid beta oxidation.

Genes involved in cholesterol biosynthesis on the other hand, such asthose encoding lanosterol synthase (Lss), sterol-C4-methyl oxidase-like(Sc4 mol), farnesyl diphosphate synthetase (Fdps), NAD(P) dependentsteroid dehydrogenase-like (Nsdhl) and3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (Hmgcs1) weredown-regulated in this study (FIG. 2B). It should be noted that the foldchanges for most of the genes in the GenMAPP were negative, indicatingdown-regulation, even for genes which were not selected as significantlydifferent based on the selection criteria used. Hmgcr which encodes for3-hydroxy-3-methylglutaryl-coenzyme-A reductase, an enzyme inhibited bycholesterol-lowering statins, showed a negative fold change as well,although the value was not significantly different.

Cholesterol is an important constituent of cellular membranes and servesas a precursor in the formation of bile acids and steroid hormones.Excessive cholesterol however, is involved in atherosclerotic lesion andgallstone formation. The results obtained suggest that cholesterolbiosynthesis in the livers of these mice was reduced, and further implythat oil palm phenolics may help to prevent atherosclerosis andcardiovascular disease. These results (up-regulated fatty acid betaoxidation and down-regulated cholesterol biosynthesis in the liver) alsosupport earlier findings that oil palm phenolics were able to improvevascular health and reduce atherosclerosis.

Gene Expression Changes in the Liver, Spleen and Heart (Atherogenic DietModule)

The number of genes significantly changed by the atherogenic diet washighest in the liver (2593 up-regulated and 451 down-regulated),followed by the spleen (990 up-regulated and 534 down-regulated) and theheart (1441 up-regulated and 991 down-regulated). The number of genessignificantly changed by oil palm phenolics was highest in the spleen(327 up-regulated and 249 down-regulated), followed by the liver (35up-regulated and 84 down-regulated) and the heart (19 up-regulated and13 down-regulated). In the latter comparison, as the heart showed theleast number of genes significantly changed (32 genes) which would notgive much information in further functional analysis, we further reducedthis stringency by filtering for significantly changed genes with a|Differential Score| of more than 13, which was equivalent to a P Valueof less than 0.05. This yielded 132 significantly changed genes in theheart (79 up-regulated and 53 down-regulated). The lists of genessignificantly changed by the atherogenic diet and oil palm phenolics inthese mouse organs, together with the fold changes, are supplemented inAdditional Files 3 and 4 respectively. The lists of GenMAPPSs and geneontologies significantly changed by the atherogenic diet and oil palmphenolics in the major organs analyzed are given in Additional Files 5and 6 respectively.

Increased Intake of Dietary Fat and Cholesterol Up-Regulated LiverRegeneration and Down-Regulated Hepatic Cholesterol Biosynthesis

Administration of the atherogenic diet increased the turnover ofmetabolites in the liver, as shown by an up-regulation of genes involvedin the generation of precursor metabolites (anabolism) and energy(catabolism). It was also evident that genes involved in fatty acid betaoxidation, the tricarboxylic acid cycle and the electron transport chainwere up-regulated, thus suggesting an increase in energy production dueto the utilization of extra fat. In addition, the turnover of livertissues was also evident, due to the up-regulation of nuclear receptorswhich stimulate hepatocyte growth such as Hnf4a (FIG. 3A) as well ascytochrome c oxidases, complement genes and caspases involved in celldeath (FIG. 3B).

Up-regulation of the fatty acid beta oxidation process would increasethe metabolism of extra fatty acids obtained from the atherogenic diet.This eventually results in an increased energy production through thetricarboxylic acid cycle and electron transport chain. When challengedwith the atherogenic diet, the liver thus adjusts its metabolicprocesses in relation to lipid metabolism and energy production.Interestingly, mitochondrial metabolism has been implicated in theproduction of free radicals and degenerative diseases. It is thuspossible that the increase in energy production caused an increase inthe production of free radicals in the liver as well, thus resulting inoxidative stress.

As a result of this oxidative insult, nuclear receptors involved intissue growth and genes involved in cell death were up-regulated, thussuggesting that the atherogenic diet triggered hepatic inflammatoryreprogramming and liver regeneration in the mice. This also explains theenlargement of livers which was observed in these animals. An example ofa nuclear receptor up-regulated is the hepatocyte nuclear factor 4-alpha(Hnf4a), which was also found to be up-regulated when ApoE3Leiden (E3L)mice (which have lipid profiles resembling those of humans) were fed anatherogenic diet. Hnf4a is central to the maintenance of hepatocytedifferentiation and is a major in vivo regulator of genes involved inthe control of lipid homeostasis. The up-regulation of this gene andother genes associated with it suggests the important role of Hnf4a inmaintaining the proper function of the liver when challenged byoxidative stress.

Among the genes involved in cell death include those encoding cytochromec oxidases belonging to the mitochondrial electron transport chain,complement genes and caspases. The up-regulation of these genes suggeststhat cell death occurred via apoptosis as a result ofcomplement-mediated cell damage. Activation of the terminal pathway ofthe complement system leads to insertion of terminal complementcomplexes (C5b-9) into the cell membrane, which may induce cytolysis.Recent data also indicate that the terminal complement pathway (C5b-9)is involved in the induction of apoptosis via a caspase-dependentpathway. Incidentally, besides being involved in the electron transportchain, cytochrome c oxidases are essential in the apoptotic process. Theup-regulation of these three groups of genes in the same network thusimplies that the atherogenic diet caused complement activation,resulting in cell death via apoptosis. Interestingly, induction of thecomplement pathway in the liver has also been associated with lesiondevelopment in atherosclerosis-prone LDL receptor-deficient (LDLr^(−/−))mice when they were fed a high-fat Western-style diet.

As expected, genes involved in cholesterol biosynthesis weredown-regulated by the atherogenic diet (FIG. 4). Plasma or serumcholesterol levels are determined by inputs from both diets and de novobiosynthesis, utilization of cholesterol especially in the liver andsteroidogenic tissues, as well as excretion of either cholesterol orbile acids. As the atherogenic diet provided dietary cholesterol whichfurther increased cholesterol levels in the blood circulation, genesinvolved in hepatic cholesterol biosynthesis were down-regulated in thisstudy. This observation is expected due to the fact that de novocholesterol biosynthesis is down-regulated when cholesterol is availablefrom dietary intake, and partly validates the microarray gene expressiondata obtained.

A Heightened Production and Turnover of Immune Cells was Caused by theAtherogenic Diet in the Spleen

The immune system has long been implicated in atherosclerosis, which iscaused by an inflammatory response. This response is mediated byendothelial cells, platelets, monocyte-derived macrophages, dendriticcells, mast cells and specific subtypes of T lymphocytes or T cells.Advanced human atheromas also contain a heterogeneous population of Tcell receptors. Some dendritic cells cluster with T cells directlywithin atherosclerotic lesions, while others migrate to lymphoid organsto activate T cells. Macrophages, endothelial cells and smooth musclecells appear to be activated based on their expression of MHC class IImolecules and numerous inflammatory products. In addition, bone-marrowcells including hematopoietic stem cells, also contribute to thepathological remodelling in atherosclerosis by differentiating intosmooth muscle cells. Non-bone marrow-derived circulating progenitorcells in the adventitia of atherosclerotic lesions might also be asource for smooth muscle cells, macrophages and endothelial cells inthese lesions, besides the migration of these cells from the tunicamedia.

In this study, genes involved in the immune response were up-regulatedby the atherogenic diet in the spleen, such as those regulated by tumournecrosis factor-alpha (Tnfa) and signal transducer and activator oftranscription 3 (Stat3) (FIG. 5A). In addition, the apoptotic processwas also found to be up-regulated. On the other hand, genesdown-regulated by the atherogenic diet include those regulated by thetumour suppressor Tp53 (FIG. 5B) and transforming growth factor-beta(Tgfb1). Tp53 is anti-proliferative while Tgfb1 is anti-inflammatory.The up-regulation of Tnfa and Stat3, coupled with the down-regulation ofTp53 and Tgfb1, suggests the up-regulation of an inflammatory responsetowards the atherogenic diet.

It is interesting to note that Stat3 was discovered because of its rolein the acute phase response, and that this is the only capacity in whichits function in vivo can be clearly ascribed to its activity as atranscription factor. Stat3 is important for hematopoietic homeostasisas it plays a critical role in mediating cellular responses involved inthe production of immature and committed hematopoietic progenitors. Inaddition, Stat3 has been implicated in many human lymphoproliferativeand myeloproliferative diseases, including multiple myeloma, non-Hodgkinlymphoma and acute myeloid leukemia, that display deregulated Stat3activation. In support of the observation that the Stat3 network wasup-regulated, the B cell receptor pathway was also up-regulated in thisstudy, further advocating the role of Stat3 in encouraging theproliferation of immune cells. Together with the up-regulation of theStat3 network, B cell receptor pathway and apoptosis, thedown-regulation of the tumour suppressor Tp53 implies that theatherogenic diet caused an increased turnover of immune cells in thespleen, and thus explains the increased production and deployment ofimmune cells in the blood circulation, which may further excerbate thein vivo inflammatory effects of the atherogenic diet in this study.

The Atherogenic Diet Triggered an Inflammatory Response in the Heart

In the heart, the atherogenic diet increased the expression of genesinvolved in fatty acid beta oxidation, proteasomal degradation, hemebiosynthesis as well as inflammation including those regulated by Tnfa,CREB (cyclic adenosine monophosphate response element binding) bindingprotein (Crebbp) and Jun oncogene which is part of activator protein-1(Ap-1) (FIG. 6A). Down-regulated genes were found to be involved inglycolysis, circadian rhythm, muscle development and anti-inflammatorynetworks such as those regulated by sirtuin 1 (Sin1) and Tgfb1 (FIG.6B).

The Jun protein forms part of the transcription factor AP-1, which ispro-inflammatory as it has been implicated in oxidative stress. Bindingsites of the redox-regulated transcription factor AP-1 are located inthe promoter region of a large variety of genes that are directlyinvolved in the pathogenesis of diseases, including atherosclerosis.Activation of Jun via Jun amino-terminal kinase (Jnk) in response tovarious forms of stress causes arterial injury and heart disease. Inaddition, heme biosynthesis was also up-regulated by the atherogenicdiet in the heart, and this suggests increased turnover of red bloodcells, most probably caused by oxidative stress brought about by thediet.

On the other hand, Tgfb has been suggested to be anti-inflammatory inatherosclerosis, as it plays an important role in the maintenance ofnormal blood vessel structure, while defects in this superfamily ofgenes have been linked to a range of cardiovascular syndromes includingloss of healthy vessel architecture and aneurysm. Microarray profilingcarried out on the aortas from apolipoprotein E-deficient (apoE^(−/−))mice on a high-fat diet compared with control C57Bl/6J and C3H miceacross time also showed a decreased expression of an isoform of Tgfb.The down-regulation of the Tgfb1 gene in this study thus implies apro-inflammatory response to the atherogenic diet in the heart.

The Unfolded Protein Response was Up-Regulated by Oil Palm Phenolics inthe Liver

In livers of mice belonging to the atherogenic diet treatment group,genes involved in the unfolded protein response were up-regulated (FIG.7) by oil palm phenolics compared to the atherogenic diet control group.Down-regulation of genes involved in endogenous antigen presentation,fatty acid metabolism, arylsulfatase activity, NADH dehydrogenase(ubiquinone) activity and oxidoreductase activity were also observed,indicating a down-regulation of the inflammatory response and energyproduction.

Up-regulated genes involved in the unfolded protein response includeHerpud1, Tra1 and Vcp. Unfolded protein response can be promoted by thebuildup of unfolded proteins in the endoplasmic reticulum andconstitutes a mechanism to reduce this burden. It acutely reducestranslation of new proteins, followed by increased expression ofchaperones to aid folding of existing proteins and enhanced eliminationof proteins that cannot be refolded. Endoplasmic reticulum stressresponsive genes have been suggested to be a protective response toprotein unfolding or protein damage resulting from cellular stresssignals. Accordingly, decreased expression of Herpud1 were reported tobe found in prostate cancer patient specimens. Thus, oil palm phenolicsmay help to reduce the amount of damaged proteins caused by theatherogenic diet in the liver and thus lessen its turnover and metabolicburden.

Another interesting gene found regulated was Keap1, which wasdown-regulated by the atherogenic diet but up-regulated by oil palmphenolics. Keap1 is an inhibitor of Nrf2, which normally sequesters Nrf2in the cytoplasm. Under oxidative stress, the cysteine residues of Keap1are oxidized and Nrf2 migrates to the nucleus to activate phase IIantioxidant enzymes. Of particular interest, KIAA0132, a human homologof Keap1, was up-regulated by tert-butylhydroquinone (tBHQ), a stronginducer of phase II detoxification enzymes via activation of theantioxidant responsive element (ARE). Putative Nrf2 binding sites in the5′-flanking region of KIAA0132 also indicate that transcription ofKIAA0132 can be increased by the transcription factor that itsequesters, and thus this feedback effect may aim to keep in balance theexpression of ARE-driven genes.

Down-regulation of genes involved in endogenous antigen presentationsuch as H2-T23, H2-T10, Cd59a and Mug1 may be a mechanism by which oilpalm phenolics reduce inflammation brought about by the atherogenicdiet. Genes involved in fatty acid metabolism were also down-regulated,including Cpt2, Pecr, Acas2, Fads2, Abcd3 and Abcg2. Fads2 encodes therate-limiting enzyme in the synthesis of long-chain polyunsaturatedfatty acids. This function includes the synthesis of arachidonic acidthat is needed for synthesis of the eicosanoid biomediators that playcentral roles in cell signalling, cardiovascular regulation,inflammation and blood coagulation.

Down-Regulation of Antigen Presentation in the Spleen Implies that OilPalm Phenolics Attenuated the Inflammatory Response

Compared to the atherogenic diet control group, genes up-regulated inspleens of mice in the atherogenic diet treatment group are thoseinvolved in carbohydrate metabolism, glucose metabolism, glutathionemetabolism as well as cytoskeleton organization and biogenesis. Genesdown-regulated by oil palm phenolics in spleens of mice are involved inantigen presentation (FIG. 8), apoptosis, B cell receptor signalling,defence response, genes specific to blood and lymph tissues, hemebiosynthesis, immune response, regulation of apoptosis, T cellactivation and differentiation as well as T cell receptor signalling.

Transketolase (Tkt), which controls the nonoxidative branch of thepentose phosphate pathway, provides NADPH for biosynthesis and reducingpower for several antioxidant systems [82]. It was up-regulated in thespleen by oil palm phenolics, together with glucose-6-phosphatedehydrogenase (X-linked) (G6pdx) and phosphogluconate dehydrogenase(Pgd), all of which are involved in the pentose phosphate pathway. Theproducts of the pentose phosphate pathway are important for thebiosynthesis of purine and for stimulating antioxidant response pathwaysin conjunction with the action of dietary phenolic antioxidants. Thismay also explain the up-regulation of antioxidant genes including Mgst1,Mgst2, Gsr and Gstm1 in the spleen by oil palm phenolics. Additionally,genes encoding stefins (Stfa1, Stfa2) were up-regulated as well. Thesecystatins are natural inhibitors of cysteine cathepsins, which have beenimplicated in antigen presentation and inflammation. In addition, Anxa2,a phospholipase inhibitor, was up-regulated. Annexin A2 is a pleiotropicprotein which has been proposed to function inside the cell in sortingof endosomes and outside the cell in anti-coagulant reactions.

Genes encoding MHC molecules such as H2-Ab1 and H2-Eb1 which have beenimplicated in atherosclerosis, were down-regulated in the spleen, thussuggesting that oil palm phenolics were able to attenuate theinflammatory response brought about by the atherogenic diet. Other MHCgenes down-regulated include H2-Aa, H2-Bf, H2-Dma, H2-DMb2, H2-Ea,H2-Q6, H2-Q7, H2-T9, H2-T10, H2-T17 and H2-T23. Activated macrophagesand smooth muscle cells express MHC II antigens such as HLA-DR thatallow them to present antigens to T cells, which cause atherosclerosis.In addition, MHC II expression is also central to the immune regulationin T cell-mediated autoimmune diseases.

The gene expression of MHC II molecules are transcriptionally regulatedby the class II transcriptional activator (CIITA or C2ta). CIITAactivates the expression of MHC II in all types of professionalantigen-presenting cells (macrophages, dendritic cells, B lymphocytes),of which dendritic cells are the most potent among the three.Interferon-γ represses collagen synthesis and increases the expressionof MHC II molecules in aortic smooth muscle cells through CIITA,contributing to atherosclerosis. In line with the down-regulation ofmajor histocompatibility complexes, the C2ta gene was down-regulated inthis study (FIG. 8). This is similar to the effects of statins, whichare largely used in the treatment of cardiovascular disease not onlybecause of their therapeutic effect in lowering cholesterol levels butalso in decreasing the expression of MHC II genes, in which C2ta hasbeen demonstrated as a target.

The Ccr7 receptor present on the surface of secondary lymphoid cells,functions to attract dendritic cells which migrate to secondary lymphoidorgans to present antigens to activate naive T cells. A mechanism ofanti-inflammation by antioxidants is through the modulation of cytokineinduction during inflammation. In line with this, cytokines and cytokinereceptors such as Ccl5, Ccl19 and Ccr7 were down-regulated by oil palmphenolics in this study. Additionally, antigenic markers such as Cd3d,Cd24a, Cd59b, Cd72, Cd79a, Cd79b, Cd83 and Cd86 were down-regulated.These markers are present on dendritic cells and interact withcounter-receptors on T cells to enhance co-stimulation and adhesion.Cd83 and Cd86 are specific markers of mature dendritic cells, which areup-regulated by oxidative stress through a NF-κB-dependent mechanism.The down-regulation of MHC II genes and genes encoding antigenic markersthus suggests that oil palm phenolics suppressed the inflammatoryresponse associated with the atherogenic diet, and this may represent amechanism by which oil palm phenolics ameliorate atherosclerosis.

Antioxidant Genes were Up-Regulated by Oil Palm Phenolics in the Heart

In hearts of mice, genes up-regulated by oil palm phenolics includethose involved in oxidative stress (FIG. 9), circadian exercise andnucleosome assembly. Down-regulated genes on the other hand, areinvolved in electron transport and signalling as well as cellproliferation and migration.

Up-regulated genes involved in antioxidant activities include Mgst1 andGpx1. These antioxidant genes are essential in the detoxification ofcarcinogens and scavenging of reactive oxygen species. Fstl1 or TSC-36,which has been shown to inhibit the proliferation of vascular smoothmuscle cells in vitro and in vivo following stimulation of TGF-β, wasup-regulated as well. Down-regulated genes on the other hand, areinvolved in electron transport and signalling. Genes involved in cellproliferation and migration (which have been implicated inatherosclerosis), such as Egf, Ltbp4, Smtn, Vtn and Lgals4 weredown-regulated as well. Alas2, a gene which is red cell specific, wasalso down-regulated. This is in contrast with the observation that theatherogenic diet up-regulated genes involved in heme biosynthesis, whichfurther indicates that oil palm phenolics decreased heme turnover causedby the atherogenic diet and thus functioned to reduce oxidative stressin the heart.

Comparison of Genes Significantly Changed by the Atherogenic Diet andOil Palm Phenolics

In order to assess how oil palm phenolics affected genes changed by theatherogenic diet, genes significantly changed by the atherogenic dietwere intersected with genes significantly changed by oil palm phenolicsin the atherogenic diet module to obtain a set of genes which weresignificantly regulated by both factors (atherogenic diet and oil palmphenolics). This comparison is given in Additional File 7. Thepercentages of genes which were differentially regulated by both factorsin terms of direction were then calculated, with the results shown inFIG. 10. A majority (>50%) of the genes regulated by oil palm phenolicsin the different organs showed a difference in direction of regulationwhen compared to the atherogenic diet. The highest percentage of changewas found in the liver while the lowest percentage of change was foundin the spleen. FIGS. 11A and 11B together provide a diagram whichcompares the fold change direction of genes significantly changed by theatherogenic diet and oil palm phenolics, using the liver as an example.

Unchanged changes are genes which show a similar direction of regulationby the atherogenic diet and oil palm phenolics while changed genesshowed an opposite direction of regulation by the two factors. Thepercentage of changed genes was calculated by dividing the amount ofgenes which changed in terms of direction of regulation with the totalnumber of genes significantly changed by both factors.

ND+DW indicates Normal Diet+Distilled Water, AD+DW indicates AtherogenicDiet+Distilled Water and AD+OPP indicates Atherogenic Diet+Oil PalmPhenolics. Values of fold changes are represented using ablue-black-yellow (negative to positive) colour scheme. The|Differential Score| for all genes is more than 20, equivalent to a PValue of less than 0.01.

Real-Time qRT-PCR Validation

To confirm the microarray results, the expression levels of eight targetgenes (Table 3) were measured using real-time quantitative reversetranscription-polymerase chain reaction (qRT-PCR). The first two targetgenes were found to be changed by oil palm phenolics in the normal dietmodule (first comparison). For the atherogenic diet module, as the focusof this study was more to identifying the changes caused by oil palmphenolics rather than the atherogenic diet, the remaining six targetgenes chosen for real-time qRT-PCR were from the third comparison(Atherogenic Diet+Oil Palm Phenolics: Atherogenic Diet+Distilled Water).The genes were chosen based on their differential scores, in which themost significantly up-regulated and down-regulated genes in each of theorgan tested were selected.

TABLE 3 Genes Selected for the Real-Time qRT-PCR Validation ExperimentsOrgan Symbol Definition Accession Assay ID Liver Cyp3a11 Mus musculuscytochrome P450, NM_007818 Mm00731567_m1 family 3, subfamily a,polypeptide 11 Liver Hmgcs1 Mus musculus 3-hydroxy-3-methyl- NM_145942Mm00524111_m1 glutaryl-Coenzyme A synthase 1 Liver Herpud1 Mus musculusubiquitin-like domain NM_022331 Mm00445600_m1 member 1 Liver Fads2 Musmusculus fatty acid desaturase 2 NM_019699 Mm00517221_m1 Spleen Anxa2Mus musculus annexin A2 NM_007585 Mm00500307_m1 Spleen Cfb Mus musculushistocompatibility 2, NM_008198 Mm00433909_m1 complement componentfactor B Heart Fstl1 Mus musculus follistatin-like 1 NM_008047Mm00433371_m1 Heart Alas2 Mus musculus aminolevulinic acid NM_009653Mm00802083_m1 synthase 2, erythroid All Sfrs9* Mus musculus splicingfactor, NM_025573 Mm00470546_m1 arginine/serine rich 9 All Guk1* Musmusculus guanylate kinase 1 NM_008193 Mm00433888_m1 All Hnrpab* Musmusculus heterogeneous nuclear NM_010448 Mm00468938_m1 ribonucleoproteinA/B *Housekeeping gene.

These genes were also present in the GenMAPPs and gene ontologiesidentified as significantly changed by the GenMAPP software. These genesalso showed detection levels of 1.0000 in both the control and treatmentgroups, which indicate that they were significantly expressed in bothgroups. In addition, the genes chosen were also present as single splicetranscripts in the microarrays used. The reason for this selectioncriterion was to minimize discordance between the two gene expressionprofiling techniques as differences in probe designs between microarraysand TaqMan assays might result in detection of additional splicevariants. Finally, all of the TaqMan assays selected had suffixes of m1,which indicate that the probes were designed across splice junctions,and would avoid the detection of genomic DNA.

Expression levels of target genes were normalized to the geometric meanof three housekeeping genes, Sfrs9, Guk1 and Hnrpab. These genes werechosen as they were shown to be stable across the previously obtainedmicroarray data. Eukaryotic 18S rRNA Endogenous Control was also testedtogether with the three housekeeping genes in preliminary experiments,and their stabilities were determined using the geNorm software.However, this gene was found to be the least stable of the fourhousekeeping genes tested and was thus further dropped as an endogenouscontrol (data not shown).

Expression fold changes for each gene quantitated by the qBase softwarebased on the real-time qRT-PCR data obtained, together with thosedetermined by the previous microarray experiments, are shown in FIG.12A. The direction and magnitude of fold changes obtained from thereal-time qRT-PCR technique were comparable to those obtained from themicroarray technique. As shown in FIG. 12B, correlation of fold changesobtained by the two gene expression profiling techniques was high(R²=0.9877), thus validating the microarray data obtained.

Serum Cytokine Profiling Supported In Vivo Anti-Inflammatory Effects ofOil Palm Phenolics

Multiplex cytokine profiling on serum samples was carried out using theBio-Plex Suspension Array System (Bio-Rad Laboratories, Hercules,Calif.), which is a microbead and flow-based protein detection systembased on the Luminex xMAP technology, available at the MedicalBiotechnology Center, Faculty of Medicine, University of Malaya. TheBio-Plex Mouse Cytokine 23-Plex Cytokine Panel (Bio-Rad Laboratories,Hercules, Calif.), which included antibody-conjugated beads for 23 typesof mouse cytokines, was also utilized. The cytokines present in thispanel include IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10,IL-12 (p40), IL-12 (p70), IL-13, IL-17, Eotaxin, G-CSF, GM-CSF, IFN-γ,KC, MCP-1 (MCAF), MIP-1α, MIP-1β, RANTES and TNF-α.

The experiment was carried out according to manufacturer's instructions.Each serum sample (n=6) was tested in duplicates. The data were analyzedusing the Bio-Plex Manager Version 4.0 software (Bio-Rad Laboratories,Hercules, Calif.). Generation of standard curves, averaging of duplicatefluorescence readings of each serum sample, background subtraction withthe blank and calculation of concentration for each cytokine werecarried out by the Bio-Plex Manager software. The averaged concentrationreadings were exported into Microsoft Excel for statistical analysis, inwhich the two-tailed unpaired Student's t-test was used. Differenceswith t-test p-values of less than 0.05 were considered statisticallysignificant.

For serum cytokine profiling, the amounts of eotaxin were surprisinglyhigh for all animals in the three groups (FIG. 13A). This may be causedby the exposure of the animals to the non-sterile environment as theywere not maintained in a specific pathogen-free facility. In the normaldiet module, RANTES was significantly reduced in the treatment groupcompared to the control group (FIG. 13B). This may be a sign of loweredinflammation as RANTES had been implied in inflammation, obesity andcerebral microvascular dysfunction. For those on the atherogenic diet,there was a significant decrease in interleukin-12 (p40 subunit) (IL-12(p40)) and a significant increase in interleukin-13 (IL-13) in the groupgiven oil palm phenolics when compared to the atherogenic control group(FIG. 13B).

As a component of the immune response, cytokines too play an importantrole in mediating the inflammatory response in atherosclerosis.Atherosclerotic lesions normally contain cytokines that promote a Th1cellular immune response (interferon-γ, interleukin-1, interleukin-2,TNF-α and TNF-β) rather than a Th2 humoral immune response(interleukin-4, interleukin-5 and interleukin-10) [99]. In micebelonging to the atherogenic diet treatment group, a decrease in thepro-inflammatory IL-12 (p40) cytokine and an increase in theanti-inflammatory IL-13 cytokine in the sera were observed when comparedto the atherogenic diet control group. This is believed to be anattenuation of the inflammatory response towards atherosclerosis.

IL-12 is a cytokine of innate immunity which is secreted by activatedmacrophages and dendritic cells, and is a key inducer of cell-mediatedimmunity as it stimulates the production of IFN-γ, stimulates thedifferentiation of CD4+ helper T lymphocytes into T_(H)1 cells as wellas enhances cytolytic functions of activated NK cells and CD8+ cytolyticT lymphocytes. It has been implicated in atherosclerosis and otherinflammatory diseases, and have been found to be attenuated by severalantioxidative plant compounds such as catechins, curcumin, apigenin andsilibinin. IL-13 is a cytokine of adaptive immunity which is secreted byCD4+ helper T lymphocytes (T_(H)2 cells), and it inhibits macrophagesand antagonizes IFN-γ. The anti-inflammatory effects observed in theserum samples were consistent with the gene expression changes seen inthe spleens of mice given oil palm phenolics, which indicate attenuationof the inflammatory response.

Serum Antioxidant Analysis Confirmed In Vivo Antioxidant Effects of OilPalm Phenolics

The basic mechanism for the antioxidant assays used in this studyinvolves the transfer of an electron from the antioxidant to the probe,which is normally an oxidant. This results in the formation of anoxidized antioxidant and a reduced probe. Antioxidant analysis on serumsamples was carried out using four assays including the total phenolicscontent by Folin-Ciocalteu reagent (TP-FCR) assay, the ferric reducingability of plasma (FRAP) assay, the 2,2-diphenyl-1-picrylhydrazyl (DPPH)scavenging activity assay and the Trolox equivalent antioxidant capacity(TEAC) assay. All these assays were carried out using the Infinite M200microplate reader (Tecan, Austria). Each serum sample (n=6) was testedin duplicates. Measurement settings and data acquisition were carriedout using the Magellan Version 6.2 software (Tecan, Austria). Generationof standard curves, averaging of duplicate absorbance readings of eachsample, background subtraction with the blank, calculation ofconcentration and statistical analysis for each assay were carried outin Microsoft Excel. Statistical analysis was carried out by using thetwo-tailed unpaired Student's t-test. Differences with t-test p-valuesof less than 0.05 were considered statistically significant.

For the TP-FCR assay, gallic acid was prepared in a range of 0 to 2000mg/mL to generate the standard curve. For serum analysis, 15 μL of 100%ethanol was added to 15 μL of each serum sample in order to precipitatemacromolecules out. The mixture was then vortexed for two minutes andcentrifuged at 1100×g for five minutes. The clear supernatant was thencollected for analysis. A master mix containing 40 μL of distilled waterand 4 μL of Folin-Ciocalteu reagent for each reaction to be carried outwas prepared. This master mix was then aliquoted into a clear 96-wellflat bottom microplate. The microplate was read at an absorbance of 765nm. 2 μL of sample or gallic acid standard diluent was then pipettedinto each well, followed by 20 μL of 15% w/v disodium carbonate(Na₂CO₃). The microplate was then shaken at maximum intensity for 10seconds and incubated at room temperature for 2 hours. Absorbance wasread at 765 nm. The ΔA765 nm and concentration of gallic acid equivalentfor each sample were calculated based on the standard curve obtained.

For the FRAP assay, ferrous sulphate heptahydrate (FeSO₄.7H₂O) wasprepared in a range of 0 to 2000 μmol/L to generate the standard curve.Solutions A, B and C were then prepared. Solution A comprised of 300 mMacetate (C₂H₃NaO₂.3H₂O) buffer pH 3.6 in 16% v/v acetic acid (C₂H₄O₂).Solution B comprised of 10 mM 2,4,6,-tri(2-pyridyl)-s-triazine (TPTZ)solution in 40 mM hydrochloric acid (HCl). Solution C comprised of 20 mMferric chloride hexahydrate (FeCl₃.6H₂O) solution in distilled water.The straw coloured FRAP reagent was then prepared by mixing 25 mL ofSolution A, 2.5 mL of Solution B and 2.5 mL of Solution C. It was thenkept in a water bath at 37° C. 180 μL of FRAP reagent was then aliquotedinto a clear 96-well flat bottom microplate. The microplate was read atan absorbance of 593 nm. 18 μL of sample or FeSO₄.7H₂O standard diluentwas then pipetted into each well. The microplate was then shaken atmaximum intensity for 10 seconds and incubated at 37° C. for 10 minutes.Absorbance was read at 593 nm. The ΔA593 nm and concentration of Troloxequivalent for each sample were calculated based on the standard curveobtained.

For the DPPH assay, Trolox was prepared in a range of 0 to 500 μmol/L togenerate the standard curve. For serum samples, 15 μL of 100% ethanolwas added to 15 μL of each serum sample in order to precipitatemacromolecules out. The mixture was then vortexed for 2 minutes andcentrifuged at 1100×g for 5 minutes. The clear supernatant was thencollected for analysis. 0.2 mmol/L DPPH was prepared in 50% v/v ethanol.95 μL of this DPPH solution was then aliquoted into a clear 96-well flatbottom microplate. The microplate was read at an absorbance of 515 nm. 5μL of sample or Trolox standard diluent was then pipetted into eachwell. The microplate was then shaken at maximum intensity for 10 secondsand incubated at room temperature for 10 minutes. Absorbance was read at515 nm. The ΔA515 nm and concentration of Trolox equivalent for eachsample were calculated based on the standard curve obtained.

For the TEAC assay, a 7 mM2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) solution anda 2.45 mM dipotassium persulfate (K₂O₈S₂) solution were first preparedin distilled water each. The ABTS reagent was then prepared by mixing 25mL of ABTS solution with 12.5 mL of K₂O₈S₂ solution and held in darknessfor 16 hours at room temperature to produce a dark green colouredsolution. The following day, Trolox was prepared in a range of 0 to 200μmol/L to generate the standard curve. 180 μL of ABTS reagent was thenaliquoted into a clear 96-well flat bottom microplate. The microplatewas read at an absorbance of 734 nm. 18 μL of sample or Trolox standarddiluent was then pipetted into each well. The microplate was then shakenat maximum intensity for 10 seconds and incubated at 37° C. for 6minutes. Absorbance was read at 734 nm. The ΔA734 nm and concentrationof Trolox equivalent for each sample were calculated based on thestandard curve obtained.

For serum antioxidant analysis, the standard curves obtained for thefour assays carried out had R² values >0.9 (data not shown). Nosignificant changes were caused by oil palm phenolics in the normal dietmodule (see FIGS. 14A-14D), and this was quite unexpected. However, itshould be noted that the administration of a commercially availablesource of olive phenolics (Olivenol Livin′) derived from olive millwastewater also did not increase plasma total antioxidant statusalthough blood was drawn one hour after ingestion of the preparation foranalysis, similar to previous studies carried out on olive oilwastewater extract and olive leaf supplements. The lack of effect on thetotal antioxidant capacity observed was interpreted in terms of lowerattainable concentrations of olive phenolics as compared to endogenousantioxidants. This may also be the case for oil palm phenolics.

The antioxidant analysis carried out on the serum samples showed thatfor the atherogenic diet control group, there was a significant decreasein antioxidant capacity when compared to mice given the normal diet,which indicates a higher oxidative stress in mice given the atherogenicdiet (see FIGS. 14A-14D). This is similar to the observations carriedout by previous studies. The atherogenic diet treatment group on theother hand, showed almost similar antioxidant capacity when compared tomice given the normal diet, thus indicating that the antioxidantresistance of mice supplemented with oil palm phenolics was still highalthough they were also given the atherogenic diet. This further impliesthat oil palm phenolics restored the antioxidant capacity of mice giventhe atherogenic diet, and is in line with the gene expression changesobserved in the major organs of mice, in which antioxidant genes wereup-regulated.

As a summary, it was found that oil palm phenolics up-regulated fattyacid beta oxidation and down-regulated cholesterol biosynthesis inlivers of mice given the normal diet. This might explain the slightdelay in weight gain caused by the extract, and further imply theapplication of oil palm phenolics in promoting weight loss andpreventing obesity. The administration of the atherogenic diet increasedcellular proliferation and turnover in the major organs of mice studied,including the liver, spleen and heart. An increased intake of fat andcholesterol caused an increased circulation and utilization of therespective metabolites in these organs, which further induced oxidativestress, inflammation, injury, cellular proliferation and tissueregeneration to compensate for the increased metabolic burden,especially in the liver. Among the genes found to be regulated by theatherogenic diet, it was most apparent that those linked to thepro-inflammatory Tnfa were up-regulated, while those linked to theanti-inflammatory Tgfb were down-regulated, especially in the spleen andheart.

On the other hand, oil palm phenolics showed signs of attenuating theeffects of the atherogenic diet. This extract increased unfolded proteinresponse in livers of mice, which is important in getting rid ofmisfolded proteins, while attenuated antigen presentation and processingin spleens of mice, similar to the effects of statins. Oil palmphenolics also increased the expression of antioxidant genes in thehearts of these mice. A majority (>50%) of the genes regulated by oilpalm phenolics in the different organs showed a difference in thedirection of regulation when compared to the atherogenic diet.

Despite that fact that oil palm phenolics did not significantly alterthe body and liver weights as well as the clinical biochemistry andhematology parameters of mice on the atherogenic diet, further cytokineprofiling and antioxidant analysis on mouse blood serum samples managedto confirm the in vivo anti-inflammatory and antioxidant effects of theextract. In contrast with the effects of oil palm phenolics whichdown-regulated cholesterol biosynthesis genes in mice fed the normaldiet, the extract did not cause a further reduction in this group ofgenes. This made sense as administration of the atherogenic diet alreadydown-regulated cholesterol biosynthesis, and thus furtherdown-regulation of the pathway would be futile to preventatherosclerosis. On the other hand, oil palm phenolics acted as ananti-inflammatory agent and an antioxidant in mice given the atherogenicdiet to prevent oxidative stress caused by the diet. These findingssuggest that oil palm phenolics can be used to overcome the effects ofan atherogenic diet and further imply the potential of this extract as achemopreventive agent for atherosclerosis and cardiovascular disease.

Generally, the composition in accordance with the present invention maybe prepared in various suitable forms for direct or oral administrationfor the health purposes as discussed earlier in the preceding sections.

For instance, the compositions of the present invention may be providedin the following forms, but no limiting to, suitable for oraladministration containing a pre-determined amount of the extract; asolution or a suspension in an aqueous or non-aqueous liquid, tablets,capsules and the likes.

The compositions of the invention may also be administered to a human ina dietary supplement form. Dietary supplements incorporating the activecomposition can be prepared by adding the composition to a food in theprocess of preparing the food. The composition is added to the food inan amount selected to deliver a desired dose of the composition to theconsumer of the food.

The composition comprising the compounds in accordance with the presentinvention may be prepared for use in a pharmaceutically effective ornutraceutically effective amount, solely on its own or in combinationwith other agents or compounds deemed appropriate by a person skilled inthe art.

In one embodiment the compositions may be administered in form of doses,within a predetermined period of time, whereby it may be administeredfor example but not limiting to daily, weekly or monthly.

In another embodiment the compositions may be provided in conventionaltreatment forms, pharmaceutical formulations or as nutritionalsupplement.

In one embodiment the composition of the present invention may beprovided in a nutraceutical form.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

The invention claimed is:
 1. A method for treating obesity and reducingcholesterol biosynthesis in a patient in need thereof, said methodcomprising administering to said patient a pharmaceutically acceptableamount of a composition comprising water-soluble oil palm phenolicsextracted from a palm oil mill effluent using a solvent-free process,wherein an antioxidant content of the oil palm phenolics is about 1500ppm gallic acid equivalent; and wherein the antioxidant content of theoil palm phenolics up-regulates fatty acid beta oxidation genes, andwherein the antioxidant content of the oil palm phenolics down regulatescholesterol biosynthesis genes.
 2. The method of claim 1, wherein thecomposition is provided in solid dosage form.
 3. The method of claim 1,wherein the composition is provided in liquid dosage form.
 4. The methodof claim 1, wherein the composition is provided in solution form.
 5. Themethod of claim 1, wherein the composition is provided in apharmaceutically effective form.
 6. The method of claim 1, wherein thecomposition is provided in nutraceutical form.
 7. The method of claim 1,wherein the composition is in nutritional supplementary form.
 8. Themethod of claim 1, wherein the composition is provided in a formsuitable for oral administration.
 9. The method of claim 1, whereinfatty acid beta oxidation genes are those encoding sterol carrierprotein (Scp), lysophospholipase (Lypla1), monoglyceride lipase (Mgll),acetyl-coA dehydrogenase (Acadl), acyl-coA dehydrogenases (Acads,Acad8), hydroxyacyl-coA dehydrogenases (Hadhb, Hadhsc), acetyl-coAacetyltransferases (Acat2, Acat3) and acetyl-coA acyltransferase(Acaa2).
 10. The method of claim 1, wherein the cholesterol biosynthesisgenes are those encoding lanosterol synthase (Lss), sterol-C4-methyloxidase-like (Sc4 mol), farnesyl diphosphate synthetase (Fdps), NAD(P)dependent steroid dehydrogenase-like (Nsdhl) and3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (Hmgcll).